CHO HCP ELISA Kit (One-step ELISA) HYI111
This kit utilizes a Fed-batch CHO (K1&S) cell culture to produce HCPs, which are used for the generation of specific antibodies via sheep immunization. The kit employs a solid-phase Enzyme-linked Immunosorbent Assay (ELISA) with a double-antibody sandwich technique to detect residual CHO host cell proteins (HCPs) in the sample. Polyclonal antibody specific to CHO HCPs was employed in the assay to capture any remaining HCPs in the sample. Both the Calibration Standard (or test sample) and the HRP (Horseradish Peroxidase) labeled anti-CHO HCP antibody were simultaneously added to the microtiter plate, which coated with the affinity purified capture antibody and followed by incubation and washing. Then TMB (3,3',5,5' -tetramethylbenzidine) substrate added for reaction, HRP catalyzed the oxidation of TMB by H2O2 to produce a blue product (maximum absorption peak at 655 nm). Then the stop solution is added to terminate the enzymatic reaction, resulting in a yellow color product (maximum absorption peak at 450nm). The absorbance values at 450 nm wavelength were positively correlated with the HCPs concentration in the Calibration Standard and the sample. The concentration of CHO HCPs in the sample can be calculated using the dose-response curve. No special treatment is required for the test sample and its suitability could be verified by the appropriate dilution ratios with the kit.
Applications
This kit is intended for use in determining the presence of host cell proteins (HCPs) in products manufactured by expression in CHO host cells, such as monoclonal antibodies, recombinant proteins, vaccines and so on. The kit is for RESEARCH USE ONLY and not intended for clinical use.
Figure 1. Schematic diagram
Components
|
Reagent |
Quantity |
Notes |
|
CHO HCP Calibration Standard |
2 bottles |
Lyophilized powder. Dissolve it with 500 μL Reconstitution Solution and let it stand for about 5 minutes. The solution should be clear and transparent. Please refer to the bottle label for details. |
|
Anti-CHO HCP Microtiter Strips |
8 well ×12 strips |
Strips pre-coated with sheep anti-CHO HCP affinity antibody in a vacuumed bag with desiccant. Seal and store immediately after use. |
|
Reconstitution Solution |
1×1.5 mL |
Only used to dissolve CHO HCP Calibration Standard. |
|
Diluent |
2×25 mL |
For dilution of Calibration Standard, Anti-CHO:HRP (100×) and samples. |
|
Wash Buffer Concentrate (10×) |
2×25 mL |
It is easy to crystallize at low temperatures, and can be dissolved in 37℃ water bath before use. Dilute at 1:10 with freshly prepared ultra-pure water for plate washing. |
|
Anti-CHO:HRP (100×) |
1×120 μL |
Affinity purified sheep antibody conjugated to HRP. Dilute 100 times before use. |
|
TMB Substrate |
1×12 mL |
Equilibrate to room temperature for 20 minutes before use. Keep away from light and sealed |
|
Stop Solution |
1×6 mL |
1 M hydrochloric acid. Avoid direct contact with eyes, skin, and clothing. |
|
Sealing Film |
3 pieces |
Cover the strips with it during incubation to prevent contamination and liquid evaporation. |
Note: Room temperature refers to 25±3℃.
Storage Conditions
Store the kit at 2-8℃. Use within the expiration date labeled upon the kit package. The opened components should be stored as follows.
Table 2. Recommended storage conditions for opened components
|
Component |
Stability |
|
Anti-CHO HCP Microtiter Strips |
Store in the bag with desiccant at 2-8℃ for up to 60 days. |
|
Reconstituted CHO HCP Calibration Standard |
Store at 2-8℃ for up to 30 days or below -20℃ and no more than 3 freeze-thaw cycles. |
Materials Required But Not Supplied in the Kit
1. Sterile centrifuge tubes for dilution
2. Absorbent paper for plate drying
3. Pipette Tips
4. Multi-channel reagent reservoirs (50 mL)
Related equipment
1. Microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 620 nm to 650 nm.
2. Single or multi-channel pipettes
3. Microplate thermoshaker
4. Incubator (optional)
5. Plate washer (optional)